Abstract：
NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from the bacterium Staphylococcus aureus (SauFDH) plays an important role in the vital activity of this bacterium, especially in the form of biofilms. Understanding its mechanism and structure-function relationship can help to find special inhibitors of this enzyme, which can be used as medicines against staphylococci. The gene encoding SauFDH was successfully cloned and expressed in our laboratory. This enzyme has the highest kcat value among the described FDHs and also has a high temperature stability compared to other enzymes of this group. That is why it can also be considered as a promising catalyst for NAD(P)H regeneration in the processes of chiral synthesis with oxidoreductases. In this work, the principle of rational design was used to improve SauFDH catalytic efficiency. After bioinformatics analysis of the amino acid sequence in combination with visualization of the enzyme structure (PDB 6TTB), 9 probable catalytically significant positions 119, 194, 196, 217-219, 246, 303 and 323 were identified, and 16 new mutant forms of SauFDH were obtained and characterized by kinetic experiments. The introduction of the mentioned substitutions in most cases leads to a decrease in stability at high temperatures and an increase at low temperatures. Substitutions in positions 119 and 194 lead to a decreasing of KMNAD+. A consistent decrease in the Michaelis constant in the Ile-Val-Ala-Gly series at position 119 of SauFDH is shown. KMNAD+ of mutant SauFDH V119G decreased by 27 times compared to the wild-type enzyme. After substitution Phe194Val KMNAD + decreased by 3.5 times. The catalytic constant for this mutant form practically did not change. For this mutant form, an increase in catalytic efficiency was demonstrated through the use of a multicomponent buffer system.
摘要：
NAD+依赖性甲酸脱氢酶(FDH，EC1.2.1.2）来自细菌金黄色葡萄球菌（SauFDH）在该细菌的重要活性中起着重要作用，尤其是生物膜的形式。了解其机制和结构-功能关系可以帮助找到该酶的特殊抑制剂，可用作抗葡萄球菌的药物。我们实验室成功克隆并表达了编码SauFDH的基因。该酶在所述FDH中具有最高的kcat值,并且与该组的其他酶相比还具有高温稳定性。这就是为什么在使用氧化还原酶的手性合成过程中，它也可以被认为是NAD（P）H再生的有前途的催化剂。在这项工作中,采用合理设计的原则提高了SauFDH催化效率。在结合酶结构可视化（PDB6TTB）对氨基酸序列进行生物信息学分析后，确定了9个可能的催化重要位置119、194、196、217-219、246、303和323，获得了16种新的SauFDH突变形式，并通过动力学实验进行了表征。在大多数情况下,所述取代的引入导致在高温下稳定性的降低和在低温下稳定性的提高。位置119和194的替换导致KMNAD+的降低。显示了SauFDH的位置119处的Ile-Val-Ala-Gly系列中的米氏常数的一致降低。与野生型酶相比，突变体SauFDHV119G的KMNAD降低了27倍。取代后Phe194ValKMNAD+下降了3.5倍。这种突变形式的催化常数实际上没有变化。对于这种突变形式，通过使用多组分缓冲系统证明了催化效率的提高。
